The project will investigate the transcriptional regulation of the gene that encodes the human immunodeficiency virus type 1 (HIV-1) restriction factor Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (APOBEC3G). An understanding of the transcriptional regulation of this gene may provide a means to increase APOBEC3G expression levels in HIV-1 target cells, thereby counteracting the antagonistic effect of the viral accessory protein Vif. If this could be done in vivo it would suppress HIV-1 replication in infected patients, reducing pathogenesis. APOBEC3G is a cytosolic cytidine deaminase1, 2 that serves as part of the host innate immune response. It restricts the infectivity of HIV-1 in the absence of Vif in primary human T cells, monocytes, macrophages and lymphocyte derived cell lines3. To prevent restriction by APOBEC3G, the viral protein Vif induces its ubiquitination and subsequent degradation in the producer cell1. In HIV-1?vif, APOBEC3G is incorporated into retroviral particles. Upon infection of the target cell, APOBEC3G triggers G-->A mutations by deaminating cytidine residues in the viral ssDNA during reverse transcription4. Inhibition of HIV-1?vif correlates with APOBEC3G expression. Cells that restrict HIV- 1?vif are termed non-permissive, whereas those that support the replication of HIV-1?vif are called permissive and lack APOBEC3G expression. The factors that govern cell-type specific expression of APOBEC3G are unknown. SA1 will characterize the regulatory mechanisms of APOBEC3G expression. Luciferase expression driven by an APOBEC3G specific promoter fragment will be used to assess APOBEC3G expression in permissive and non-permissive T cell lines. The promoter fragment will be dissected to identify novel elements and transcription factors that result in transcriptional activation or repression. SA2 will identify the transcription factors that govern the differential expression of APOBEC3G. Gene expression levels of transcriptional regulators will be compared between non-permissive and permissive T cell lines by microarray analyses. Candidates will be evaluated for their effect on APOBEC3G expression by overexpression and shRNA mediated knock-down analyses. Additionally, a retroviral macrophage-derived cDNA expression library will be screened to identify a cellular factor that enables APOBEC3G expression in permissive cells. PUBLIC HEALTH RELEVANCE: AIDS remains one of the major modern-day public health problems. Current anti-retroviral medications have successfully reduced mortality and extended the lifespan of patients, but are expensive, prone to side effects, and result in drug-resistant viruses. We propose to explore an alternative strategy to reduce virus replication in patients in which an enzyme, produced in human cells that potently blocks virus replication, is expressed at higher levels in infected cells.